Autophagy, apoptosis and necrosis are key players in cell death and play an important role in many human diseases. Strategies to regulate relevant pathways have been successfully applied to the treatment of a variety of diseases [Kepp, O., et al., Nat Rev Drug Discov, 2011.10 (3): p. 221-37]. However, the relevance between autophagy and apoptosis has not been fully explained. This is due to the absence of highly accurate tools or methods that can specifically distinguish intracellular phenomena that are complexly interrelated.
Various methods have been used to measure autophagy at cellular and biological levels. Specifically, the conversion of LC3 (LC3 conversion), LC3-II puncta formation and measurement of autophagic flux are currently widely used methods for autophagy detection.
The measurement of LC3 conversion detects the conversion of LC3-I to LC3-II using immunoblotting, and LC3-II itself has a problem that it is difficult to quantify the relative amount due to decomposition by self-digestion. And the sensitivity of the LC3-II antibody is much higher than that of LC3-I, making it impossible to compare the relative amounts between the two.
The LC3-II puncta formation is measured by artificially expressing the GFP-LC3 fusion protein by transfection into cells to observe the fluorescence dot shape observed when the autophagosome is formed. This method has a disadvantage in that it is difficult to distinguish the autophagic activated cell from the normal cell because a considerable part of the dot shape is observed in the normal cells as well as the cells in which the self-digestion occurs. In addition, there are disadvantages in that overexpression of GFP-LC3 protein expressed in the cells generates fluorescence points in normal cells regardless of autophagy and GFP-LC3 protein expression itself induces self-digestion (Mizushima N. et al., Cell 2010, 140, 313-326.). Moreover, the labeling ability of GFP-LC3 protein is remarkably decreased in autolysosome produced after fusion with lysosome.
There is a method of measuring autophagic flux by treating cells with chloroquine or the like which is a lysosomal inhibitor. However, the chloroquine has been shown to induce cell death caused by apoptosis in the cells to be tested (Chuandong Fan et al., Bioorganic & amp; Medicinal Chemistry, 2006, Volume 14, Issue 9, pp. 3218-3222).
In addition, monodanysylcadaverine (MDC), a fluorescent dye, is widely used in the staining of autophagic vacuoles, but its specificity remains unclear. More importantly, most of the methods described above are used to measure autophagy only in vitro or ex vivo imaging.
In vivo imaging of autophagy has been gained to some extent by methods using transgenic mice that are presently systemically expressing LC3 fused mainly with GFP (Tian, F F, et al., 2010; Mizushima, N., et al., 2004). Another method is to inject MDC and chloroquine to measure autophagic flux in the myocardium. However, there is no complete method for monitoring autophagy in vivo (Mizushima, N., et al., 2010).
Thus, there is a need in the art for universal molecular probes that can be used both in vitro and in vivo as well as accessible and permeable to a variety of tissues in molecular imaging of autophagy.